The analysis of fatty acid-containing compounds requires previously their hydrolysis or
saponification, the separation of the non-acidic constituents (when necessary)
and the liberation of the acids from the mixture of soap.

Frequently, the lipids are transesterified to produce esters in a single step.
Alkaline hydrolysis of glycerides
The process which involves the production of soap is commonly referred to as
saponification.
Discovered and described in detail by Chevreul in 1823, the process is yet run
nearly in its original form. In his famous work, Hilditch (1956) recommends for
most natural fats saponifying 100 parts by weight of fat with a solution of 30
parts of KOH in 500 parts of alcohol (95-100%), boiling under reflux for 3
hours, followed by removal of most of the alcohol by distillation. Ethanol is
generally satisfactory but other solvents have been recommended. A small amount
of water must be present to affect rapid and complete saponification of
glyceride oils.
Henriques R (Z angew Chem 1895, 8, 721; 1896, 9, 221) reported that fats
can be saponified readily at room temperature. The process may be carried out by
dissolving 260 g of KOH in 250 ml water. After some minutes, a liter of the oil
to be saponified is added to this solution. Ethanol (10 ml) is then added. This
process was applied to fats containing easily altered fatty acids (i.e.
conjugated fatty acids, highly unsaturated).
Separation of unsaponifiable matter
After saponification, approximately half of the alcohol is removed under vacuum,
the residue being diluted with water and the unsaponifiable matter
(hydrocarbons, tocopherols, sterols, etc ) extracted by shaking with an organic
solvent, diethyl ether or petroleum ether.
Recovery of acids from soaps
Fatty acids are liberated by the addition of mineral acid, usually a 10% excess
of sulfuric acid or HCl. When very short-chain acids are present, the alcohol is
removed prior acidification under vacuum with the addition of sufficient water
to keep soaps in solution. If the amount of acids is important, the long-chain
acids are separated from the aqueous medium by allowing the mixture to stand in
the cold.

Hydrolysis of waxes
Many waxes can be saponified by dissolving 2-5 g of wax in 30 ml of alcohol and
adding 50 ml of 0.5 M KOH and refluxing the mixture for 1 or more hours. For
some wax (carnauba wax) it was recommended to use xylene instead of ethanol.

After their separation, fatty acids
may be purified by one of these procedures :

Fractional distillation at reduced pressures was
initially used to separate mixtures of fatty acids and esters derived from
natural fats. The early distillation apparatus were not very efficient. Thus,
natural mixtures were first separated into chemically similar groups prior to
distillation.

The oldest record of a distillation process dates about 3600 B.C.. It was found
at Tape Gowra in Mesopotamia, the instrument was 48 cm high and 53 cm in largest
diameter. The distillation pot had a capacity of about 40 liters and the
distillate collecting ring held about 2 liters. It was probably used to make
perfumes (Great Chemists, E Farber ed, Interscience, NY 1961). As a
science, distillation and fractional distillation had to await the discovery of
the physical laws of Dalton (1766-1844) and Raoult (1830-1901). After that, the
science of distillation developed at an amazing rate.

Distillation apparatus made its appearance in the chemical laboratory in the
early part of the 19th century, according to Underwood AJV ( Trans
Inst Chem Engrs (London) 1932, 10, 112-152). An anonymous publication (Ind
Eng Chem, News Ed 1935, 13, 140) attributes the invention of the
distillation column to Cellier-Blumenthal and Derosene in France; Thorpe JF (Thorpe's
Dictionary of appl Chem, vol 1, 1937) attributes it to Coffey in England.
These bubble-plate towers were used primarily for the commercial distillation of
spirits. An early laboratory distillation unit used for the fractionation of
fatty acid esters is illustrated in Reilly's book (Distillation, Methuen,
London, 1936). A similar instrument and its operation are described by
Hilditch (The constitution of natural fats, 1940, pp374-6).The earliest
laboratory column were simply open tubes and spiral-type column was introduced
by Warren in 1864 (Mem Am Acad Arts Sci, 1867, 9, 121). The period
1900-1930 was one during which marked advances were made. One of the most
important was the development of highly efficient packed columns. Columns
containing rotating fractionation sections were described later by Podbielniak
in 1935. This device was modified with a thin metal strip rotates at high speeds
(1000-2500 rpm) in the glass column (spinning-band column). Baker (Ind Eng
Chem, Anal Ed 1940, 12, 468) described a 18-ft column, 0.6 cm in diameter
with a separation efficiency equivalent to 70 theoretical plates. The head
pressure was 1 mm Hg. This device was used by Privett OS et al (J Am Oil Chem
Soc 1959, 36, 443) to distill several fractions of methyl esters of pork
liver lipid. One sample of esters was separated into 38 distillate fractions
with chain lengths from C14 to C22. 19 specific fatty acids could be identified
and quantified (amounts from 0.2 to 35.4% plus traces of 8 other fatty acids,
C10-C15, C17 and C19.

Saturated and unsaturated fatty acids form salts with metallic ions, whose
solubilities in water and organic solvents vary with the nature of the metallic
ion and the chain length, degree of unsaturation, and other characteristics of
the acid radicals.

The oldest and most widely used method of metallic salt separation depends on
the differential solubility of the lead salts or soaps of fatty acids in ether.
This method was introduced by Gusserow CA in 1828 (Arch Pharm 1828, 27, 153)
and improved later by Varrentrapp F (Ann Chem Liebigs 1840, 35, 196).

One of the more important modifications was
introduced by Twitchell (Ind Eng Chem 1921, 13, 806) who substituted
ethanol for diethyl ether. Both methods were used for analytical purposes but
they are preferable for preparative purpose. The solid or saturated acids are
regenerated from the insoluble lead salts by boiling the latter with dilute HCl.
After cooling, the cake of solid acids is separated and the aqueous layer is
extracted with ether. Complete and sharp separation between saturated and
unsaturated acids is impossible. The method is not reliable for the separation
of mixtures containing unsaturated acids of chain length greater than C18 or
saturated acids of chain length of C14 or shorter. It may be said that the
method is applicable with vegetable oils, but not with the seed fats of the Palmae, the rapid drying oils, the cruciferous oils, castor oil, marine oils,
and oils with trans-fatty acids.

We give below the procedure of "The
precipitation of solid fatty acids with lead acetate in alcoholic solution"
as it was written by Twitchell in 1921:

Analytical
process in detail

Weigh in a beaker as
much of the fatty acid as is estimated to contain 1 to 1.5 g. of solid
acids. In the case of a very liquid oil this amount will be about 10 g.,
while in the case of tallow it will be only 2 or 3 g. Dissolve in 95 per
cent alcohol. Dissolve 1.5 g. of lead acetate in 95 per cent alcohol. The
total alcohol for the two solutions should be about 100 cc. Heat both
solutions to boiling and pour the lead acetate solution into the solution
of fatty acid. Allow to cool slowly to room temperature, and then for
several hours, preferably over night, to about 15°C. Filter and test the
filtrate for lead with a few drops of an alcoholic solution of sulfuric
acid. If there is no precipitate, showing that lead is not in excess, the
analysis must be repeated, using less fatty acid or more lead
acetate. Wash the precipitate with 95 per cent alcohol until a sample of
the washings diluted with water remains clear. Transfer and wash the
precipitate from the filter back into the beaker using about 100 cc of 95
per cent alcohol. Add 0.5 g. of acetic acid and heat to boiling. The
precipitate will slowly dissolve. Allow to cool to room temperature and
then to 15°C. as before. Filter and wash with 95 per cent alcohol as
before. Transfer the precipitate by washing the filter paper with ether
into the beaker. Add sufficient dilute nitric acid to decompose the lead
salts. Pour and wash the whole mixture into a separatory funnel and
shake. Wash with water until the washings are no longer acid to methyl
orange. If a trace of nitric acid should remain with the ethereal solution
it will act on the fatty acids in the subsequent drying. Transfer the
ethereal solution to an evaporating dish, evaporate, dry, and weigh.

LOW-TEMPERATURE
CRYSTALLISATION

Crystallization of acids and esters from solvents at low temperatures was
introduced in the late 1930's at the time that precise fractional distillation
was being developed. It was widely applied for the separation of fatty acids and
monoesters, and also for the separation of glycerides and other lipid
substances. A review can be found from Brown JB et al. (Prog Chem fats and
other lipids, vol 3 Pergamon 1955, pp 57-94) who first applied
low-temperature crystallization for the separation of fatty acids.

Hilditch used extensively this method to fractionate glycerides of natural fats.
Various modifications were made with respect to solvents, concentration of fats
or glycerides, and temperatures. Low-temperature crystallization has been
applied for about 25 years and as an alternative to the lead salt method. It was
useful to prepare concentrates of specific unsaturated fatty acids from raw
stocks.
A review can be found from Markley KS (Fatty acids, Markley ed., Interscience
Pub, 1964, part 3, 1983-2123).

LIQUID CHROMATOGRAPHY

Michael Tswett (1872-1920) is credited with developing the first concepts and
techniques of chromatography as they are now known. Thus, the separation of
plant pigments gave rise to the first chromatographic studies. Tswett was aware
that his results possessed significance beyond the mere separation of pigments,
as is evident from his first communication. Chemists were not attracted by
methods which enable to handle only milligrams of materials.

Kuhn R et al. (Z Physiol Chem 1931, 197, 141)
rediscovered chromatography in the course of studies on carotenoids. The
subsequent applications of chromatography with fatty acids and lipids in general
will wait until 1950 to find some references in this field.

The reversed polarity partition chromatography was first used by Boldingh J (Experientia
1948, 4, 270) to separate fatty acids on sheets of filter paper impregnated
with latex to support the non-polar phase. Powdered latex was later used for
column partition chromatography (Boldingh J, Rec Trav Chim 1950, 69, 247),
and aqueous acetone or methanol as mobile phase to separate long-chain fatty
acids. Besides rubber, or glyceride oils, silicone oil or synthetic polymers
were used in paper and column chromatography.

The first separation using reversed phase column was reported by Boldingh J (Rec
Trav Chim 1950, 69, 247) who used latex powder saturated with benzene or
peanut oil as the stationary phases and methanol + acetone or methanol + water as
mobile solvents.

GAS-LIQUID
CHROMATOGRAPHY

The history of the gas-liquid chromatography of fatty acids dates from 1952,
when James AT et al. (Biochem J 1952, 50, 679) published a description of
separations of free fatty acids from 1 to 12 carbon atoms by this technique.
Later, they discovered that a previous methylation of fatty acids improved their
volatility and their separation at temperatures below 250°C (James AT et
al., Biochem J 1956, 63, 144-152). These classic papers contain yet valid
descriptions of the principles of gas chromatography and may serve as an
introduction for those needing basic information on that important technique. It
is noticeable that the first publication of James and Martin was not only the
first description of gas-liquid chromatography of fatty acids, but corresponds
also to the first description of that technique. These authors used Apiezon
stopcock grease (silicon-free hydrocarbon grease) as the stationary phase incorporated into a
solid powdered support (Celite 545) and packed into a glass column. At that time
it was not possible to analyze a complex mixture since the use of increasing
temperatures was foreseen but impossible to apply with the detector included in
the instruments. A major advance was the introduction by Orr CA et al. (J Am
Chem Soc 1958, 80, 249) of a liquid polyester phase as stationary phase able
to conveniently separate saturated and unsaturated fatty acids. Later, the use
of capillary columns with very thin films of various polar phases enabled to
profundly improve fatty acid analyses (Horning EC et al., Anal Chem 1963, 35,
526).

UREA-FATTY ACID COMPLEXES

The first description of the urea-fatty acid complexes by Bengen F in 1940 (German Patent Appl. OZ 12438, march 18, 1940) revolutionized fat chemistry for
about 20 years. For a long time this technique was efficient in preparing
relatively purified fatty acids from fats and vegetal oils. During his research
on milk, Bengen showed that urea forms in water, methanol, and ethanol
well-defined crystalline inclusion compounds with straight-chain compounds but
not with branched-chain or cyclic compounds (Bengen F, Experientia 1949, 5,
200). He generalized this phenomenon to
hydrocarbons, fatty acids, esters, alcohols, aldehydes, and ketones. In addition
to Bengen's group, several researches were conducted by petroleum companies on
the separation of linear and branched-chain hydrocarbons. These
inclusion compounds are combinations of two or more molecules, one of which is
contained within the crystalline framework of the other. The stability of urea
complexes increases with increasing chain length. Furthermore, it was observed
that the greater the degree of unsaturation the greater the deviation in
behavior from the corresponding normal saturated compound. Thus, at a constant
chain length, saturated fatty acids form urea complexes preferentially to
monounsaturated, mono- preferentially to di-unsaturated, and so on. With this
approach, oleic (Swern D et al., J Am Oil Chem Soc 1952, 29, 614),
linoleic and linolenic acids (Swern D et al., J Am Oil Chem Soc 1953, 30, 5)
were isolated and purified (80-95%) from natural sources on a Kg basis.

As an example, we give below a brief description of the procedure used by Swern and
co-workers for preparing linoleic acid from a natural oil.

To a solution of 880 g of urea in 2.7 l of hot methanol, 1 Kg of safflower oil
acids (containing 78 % linoleic acid) is rapidly added and the mixture is heated
and stirred until complete dissolution. After cooling overnight at room
temperature, the precipitate is filtered off and discarded. The filtrate is
evaporated and several volumes of warm water containing 30 ml of 6 M HCl is
added. The insoluble oil is dissolved in hexane and washed several times with
water to remove urea. Evaporation of the solvent yields 790 g of linoleic acid.
The authors distillated under vacuum this oil and obtained 610 g of 95% linoleic
acid (and 5 % oleic acid).

A simple procedure which can be easily used at a laboratory level is described
in this site.

The formation of urea complexes was also used to reduce the free fatty acid
content of natural glycerides to 1 % or less. Moreover, Schlenk H et al. (J
Am Chem Soc 1950, 72, 5001) were the first to separate unoxidized from
oxidized fatty acids by precipitation of the former as urea complexes.
Similarly, oxidatively produced polymers may be separated as they do not form
urea complexes. Abu-Nasr AM et al. (J Am Oil Chem Soc 1954, 31, 16)
applied the same process to enrich several fish oils in polyunsaturated fatty
acids. This was the basis of the purification of methyl eicosapentaenoate and
docosahexaenoate. Extensive data from the literature can be found in the
Schlenk's review (Progress in the chemistry of fats and other lipids, Vol 2,
Pergamon Press, NY, 1954, pp. 243-267)

As before the development of gas-liquid chromatography (1960) the chemical
identification of long-chain fatty acids remained a complex problem, it is
worthy of note that the formation of urea complexes and their dissociation
temperature were used to define the length of the carbon chain (Knight HB et
al. Anal Chem 1952, 24, 1331). A review on the physical description of urea
inclusion compounds may be found in the paper of Hayes DG (Inform 2002, 13,
781).